Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer

Purpose  Ovarian cancer patients with intra-tumoral CD3⁺ T-lymphocytes in primary tumor tissue have a better prognosis. This study aims to analyze the presence and relative influence of three important T-lymphocyte subsets, tumor-infiltrating CD8⁺ cytotoxic T-lymphocytes (CTL), CD45R0⁺ memory T-lymphocytes, and FoxP3⁺ regulatory T-lymphocytes (Treg), in primary tumor tissue and omental metastases of patients with ovarian cancer. Experimental design  The number of CD8⁺, CD45R0⁺, and FoxP3⁺ T-lymphocytes was determined by immunohistochemistry on a tissue micro array containing ovarian tumor tissue and/or omental metastases obtained at primary debulking surgery from 306 FIGO stage I–IV ovarian cancer patients. Immunohistochemistry data were correlated to clinicopathological parameters and survival data. Results  High number of CD8⁺ CTL and a high CD8⁺/FoxP3⁺ ratio in ovarian-derived tumor tissue were associated with increased disease-specific survival and proved to be independent prognostic factors in multivariate analyses. In advanced stage patients, the presence of CD8⁺ CTL, CD45R0⁺ memory T-lymphocytes, FoxP3⁺ Treg or a high CD8⁺/FoxP3⁺ ratio in ovarian-derived tumor tissue was associated with an increased disease specific survival in univariate analysis, as was the presence of CD45R0⁺ memory T-lymphocytes and FoxP3⁺ Treg in omental metastases. Furthermore, in advanced stage patients CD8⁺ cytotoxic and FoxP3⁺ regulatory T-lymphocytes infiltrating ovarian-derived tumor tissue were independent predictors of increased prognosis. Conclusions  T-lymphocytes infiltrating primary and metastatic ovarian cancer sites are associated with improved prognosis. These associations are especially distinct in advanced stage patients, underlining the potential for immunotherapy as a broadly applicable therapeutic strategy. Ninke Leffers and Marloes J. M. Gooden contributed equally.     

Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer

The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 ⁺ and CD4 ⁺ T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 ⁺ and CD8 ⁺ T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.     

Human kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8⁺ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201⁺ KLK4 ⁺ cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.     

Adoptive transfer of cytotoxic T lymphocytes induced by CD86-transfected tumor cells suppresses multi-organ metastases of C1300 neuroblastoma in mice

 In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86⁺C1300, expressing a high level of mouse CD86 on the cell surface. While CD86⁺C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86⁺C1300-rejecting mice with CD86⁺C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86⁺C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86⁺C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86⁺C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors. Received: 18 November 1996 / Accepted: 3 March 1997     

Defining MHC class II T helper epitopes for WT1 tumor antigen

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4⁺ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1_124–138 and WT1_247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.     

HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy

Tolerance to tumor-nonmutated self proteins represents a major obstacle for successful cancer immunotherapy. Since this tolerance primarily concerns dominant epitopes, we hypothesized that targeting cryptic epitopes that have a low affinity for HLA could be an efficient strategy to breach the tolerance to tumor Ags. Using the P1Y heteroclitic peptide approach, we identified low affinity cryptic HLA-A*0201-restricted epitopes derived from two widely expressed tumor Ags, HER-2/neu and hTERT. The P1Y variants of four HER-2/neu (neu(391), neu(402), neu(466), neu(650))- and two hTERT (hTERT(572) and hTERT(988))-derived low affinity peptides exhibited strong affinity for HLA-A*0201 and stimulated specific CTL from healthy donor PBMCs. These CTL specifically recognized HER-2/neu- and hTERT-expressing tumor cells of various histological origins. In vivo studies showed that HLA-A*0201 transgenic HHD mice vaccinated with the P1Y variant peptides generated CTL that specifically lysed Ag-expressing tumor cells, thus recognizing the cognate endogenous Ags. These results suggest that heteroclitic variants of low affinity, cryptic epitopes of widely expressed tumor Ags may serve as valid tools for tumor immunotherapy.     

An HLA-A2 polyepitope vaccine for melanoma immunotherapy.

Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. Here we describe a recombinant poxvirus vaccine that codes for ten HLA-A2-restricted epitopes derived from five melanoma Ags conjoined in an artificial polyepitope or polytope construct. Target cells infected with the melanoma polytope vaccinia were recognized by three different epitope-specific CTL lines derived from HLA-A2 melanoma patients, and CTL responses to seven of the epitopes were generated in at least one of six HLA-A2-transgenic mice immunized with the construct. CTL lines derived from vaccinated transgenic mice were also able to kill melanoma cells in vitro. Multiple epitopes within the polytope construct were therefore shown to be individually immunogenic, illustrating the feasibility of the polytope approach for melanoma immunotherapy. Tumor escape from CTL surveillance, through down regulation of individual tumor Ags and MHC alleles, might be overcome by polytope vaccines, which simultaneously target multiple cancer Ags.     

T cell precursor frequency differentially affects CTL responses under different immune conditions

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8⁺ CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4⁺ and CD8⁺ T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia^b−/− gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC_OVA)-stimulated CD8⁺ CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8⁺ T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4⁺ T cell help, and (iii) increasing CD4⁺ and CD8⁺ T cell precursors overcomes immune suppression to DC_OVA-stimulated CD8⁺ CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.     

Induction of preferential cytotoxicity against allogeneic mouse lymphoma cells: in vitro and in vivo studies

The aim of this study was to activate, in mixed leukocyte/tumor cell cultures (MLTC), cytotoxic lymphocytes exhibiting preferential activity in vitro and in vivo towards allogeneic mouse lymphoma cells. Whereas the lymphoma target cells were readily lysed by the MLTC-derived lymphocytes, the cytotoxicity against the corresponding allogeneic concanavalin-A(ConA)-induced lymphoblasts was more than tenfold lower. Both activities were mediated by CD3⁺, TCR⁺, CD8⁺, CD4⁻ cytotoxic T cells (CTL). ConA-induced lymphoblasts were readily lysed by anti-Thy1.2 antibodies and complement, by CTL derived from mixed leukocyte cultures (MLC) and by the MLTC-derived CTL in the presence of ConA, indicating that the lymphoblasts are not merely less lysable than the lymphoma cells but that the latter are specifically recognized by the CTL. Lymphoblasts poorly competed with ⁵¹Cr-labeled lymphoma cells in a “cold”-target competition assay, suggesting that the MLTC-derived CTL largely recognize epitopes expressed only by the lymphoma cells. Furthermore, analysis of the cytotoxic activity of more than 500 MLTC-derived CTL oligoclones and over 30 clones revealed that one-third of them were cytotoxic only against the allogeneic lymphoma cells, one-third were reactive against both the lymphoma and the allogeneic lymphoblast target cells and the remainder were not cytotoxic at all. Upon injection into sublethally irradiated, lymphoma-bearing allogeneic mice, the MLTC-derived CTL cured 56% of the recipients and caused graft versus host disease (GVHD) is only 22%, whereas CTL activated in MLC against allogeneic splenocytes were therapeutically ineffective and caused lethal GVHD in 89% of the recipients. Although the therapeutic efficacy of the in vitro-generated antitumor CTL was demonstrated against experimental lymphoma lines, this strategy might prove effective in tumor immunotherapy in conjunction with other modalities. Received: 24 December 1998 / Accepted: 5 April 1999     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

Many studies have demonstrated that prostate stem cell antigen (PSCA) is an attractive target for immunotherapy based on its overexpression in prostate tumor tissue, especially in some metastatic tissues. In this study, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse models. We showed that DCLV-PSCA could preferentially deliver the PSCA antigen gene to DC-SIGN-expressing 293T cells and bone marrow-derived DCs (BMDCs). Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8⁺ and CD4⁺ T cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was observed in a therapeutic model. This DCLV-PSCA vaccine also showed efficacy in inhibiting tumor metastases using a PSCA-expressing B16-F10 model. Taken together, these data suggest that DCLV is a potent vaccine carrier for PSCA in delivering anti-prostate cancer immunity.     

DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells

Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133⁺ murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133⁺ tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133⁺ tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4⁺ T cells produced CD133⁺ tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.     

Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4⁺ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as melanoma-specific. Optimal demonstration of the lytic activity of CD4⁺ CTL required a 16-h incubation period and an effectortarget cell ratio of 401. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN) consistently augmented lysis by these CD4⁺ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4⁺ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFN treatment. Thus, IFN may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4⁺CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8⁺ counterparts.This work was supported by USPHS grant R01-CA 36233, and a grant from the Concern Foundation for Cancer Research.     

In Silico Analysis of Synaptonemal Complex Protein 1 (SYCP1) and Acrosin Binding Protein (ACRBP) Antigens to Design Novel Multiepitope Peptide Cancer Vaccine Against Breast Cancer

Multiepitope cancer vaccines are manifesting as the future of breast cancer immunotherapy. In the present study, high immunogenic fragments of synaptonemal complex protein 1 (SYCP1) and acrosin binding protein (ACRBP) antigens were selected according to MHCs binding affinity and CD8⁺ cytotoxic T lymphocytes’ (CTL) epitopes by various immunoinformatic servers. (RCQHKIAEMVALMEKHKHQYDKI) residue from p702–724 SYCP1 and the (YIQYPNYCSFKSQQCL) residue from p481–496 ACRBP were selected as the immunodominant fragments. Then, the selected fragments were fused together with a furin-sensitive linker to form the final peptide vaccine construct. The cleavage sites, TAP transport efficiency, CTL epitopes, post-translational modifications, and B cells epitopes were predicted for the final construct. The final construct contained appropriate CTLs epitopes and also, several linear and conformational B cell epitopes. Also, it exhibited 90.37% HLA population coverage in the world. Therefore, these preliminary results require validation by in vitro and in vivo experiments.

     

An MVA vaccine overcomes tolerance to human p53 in mice and humans

Background The cellular regulatory protein p53 is overexpressed by almost 50% of all malignancies making it an attractive target for a vaccine approach to cancer. A number of immunotherapy approaches targeting p53 have been evaluated successfully in murine models, but translation of these preclinical findings to the clinic has been unsuccessful. Prior studies in our laboratory employing murine models demonstrated that a modified vaccinia virus Ankara (MVA) vaccine expressing murine p53 could stimulate p53 specific immunity. Systemic administration of the MVA vaccine was able to effect the rejection of established tumors. To better understand the immunologic mechanisms that underlie the vaccine function of human p53, we utilized a murine model in which the murine germ line copy of p53 was replaced with a modified human one. These mice, referred to as Hupki, were evaluated as a tolerant model to explore the capacity of MVA expressing human p53 to overcome tolerance and reject human p53-expressing tumors. Results MVAp53 immunization of Hupki mice resulted in the generation of p53-specific CD8⁺ T cells and the rejection of a highly aggressive murine mammary carcinoma cell line 4T1(H-2d) transfected with human p53 (4T1p53). An immunologic correlate of tumor protection was evaluated utilizing an overlapping peptide library spanning the full length of human p53. This reagent was also used in combination with MVAp53 to stimulate p53-specific CD8⁺ T cell responses in cancer patients. Conclusion These studies demonstrate the potential of MVAp53 to overcome tolerance to p53 for cancer immunotherapy.     

Identification of a WT1 protein‐derived peptide,WT1187, as a HLA‐A*0206‐restricted,WT1‐specific CTL epitope

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1‐specific CTL epitopes with a restriction of HLA‐A*2402 or HLA‐A*0201 have been already identified. In the present study it has been demonstrated that a 9‐mer WT1‐derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1‐specific CTL response with a restriction of HLA‐A*0201, can also elicit a CTL response with a restriction of HLA‐A*0206. In all three different HLA‐A*0206⁺ healthy donors examined, WT1₁₈₇ peptide‐specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA‐A*0206 molecules. The present study describes the first identification of a HLA‐A*0206‐restricted, WT1‐specific CTL epitope. The present results should help to broaden the application of WT1 peptide‐based immunotherapy from only HLA‐A*0201‐positive to HLA‐A*0206‐positive cancer patients as well.     

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I–restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8⁺ splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8⁺ tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8⁺ TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8⁺ splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8⁺ TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8⁺ anti-tumor response.